Immunomodulatory effects of Isochrysis galbana incorporated diet on Oreochromis sp. (red hybrid tilapia) via Sera-1H NMR metabolomics study.

Tilapia is one of the most common fish species that is intensively produced all over the world. However, significant measures at improving aquaculture health must be taken since disease outbreaks are often encountered in the rapidly developing aquaculture industry. Therefore, the objective of the study was designed to evaluate the metabolite changes in tilapia' sera through 1H NMR metabolomics in identifying the potential biomarkers responsible for immunomodulatory effect by the indigenous species of Malaysian microalgae Isochrysis galbana (IG). The results showed that IG-incorporated diet mainly at 5.0% has improved the immune response of innate immunity as observed in serum bactericidal activity (SBA) and serum lysozyme activity (SLA). The orthogonal partial least squares (OPLS) analysis indicated 5 important metabolites significantly upregulated namely as ethanol, lipoprotein, lipid, α-glucose and unsaturated fatty acid (UFA) in the 5.0% IG-incorporated diet compared to control. In conclusion, this study had successfully determined IG in improving aquaculture health through its potential use as an immune modulator. This work also demonstrated the effective use of metabolomics approach in the development of alternative nutritious diet from microalgae species to boost fish health in fulfilling the aquaculture's long-term goals.

Embryonic mercury exposure in zebrafish: Alteration of metabolites and gene expression, related to visual and behavioral impairments.

The widespread presence of mercury, a heavy metal found in the environment and used in numerous industries and domestic, raises concerns about its potential impact on human health. Nevertheless, the adverse effects of this environmental toxicant at low concentrations are often underestimated. There are emerging studies showing that accumulation of mercury in the eye may contribute to visual impairment and a comorbidity between autism spectrum disorders (ASD) trait and visual impairment. However, the underlying mechanism of visual impairment in humans and rodents is challenging. In response to this issue, zebrafish larvae with a cone-dominated retinal visual system were exposed to 100 nM mercury chloride (HgCl2), according to our previous study, followed by light-dark stimulation, a social assay, and color preference to examine the functionality of the visual system in relation to ASD-like behavior. Exposure of embryos to HgCl2 from gastrulation to hatching increased locomotor activity in the dark, reduced shoaling and exploratory behavior, and impaired color preference. Defects in microridges as the first barrier may serve as primary tools for HgCl2 toxicity affecting vision. Depletion of polyunsaturated fatty acids (PUFAs), linoleic acid, arachidonic acid (ARA), alpha-linoleic acid, docosahexaenoic acid (DHA), stearic acid, L-phenylalanine, isoleucine, L-lysine, and N-acetylputrescine, along with the increase of gamma-aminobutyric acid (GABA), sphingosine-1-phosphate, and citrulline assayed by liquid chromatography-mass spectrometry (LC-MS) suggest that these metabolites serve as biomarkers of retinal impairments that affect vision and behavior. Although suppression of adsl, shank3a, tsc1b, and nrxn1a gene expression was observed, among these tsc1b showed more positive correlation with ASD. Collectively, these results contribute new insights into the possible mechanism of mercury toxicity give rise to visual, cognitive, and social deficits in zebrafish.

Phenolic content of Terminalia catappa L. leaf and toxicity evaluation on red hybrid tilapia (Oreochromis sp.).

Despite the use of Terminalia catappa (TC) leaf by traditional fish farmers around the world to improve the health status of cultured fish, there is a paucity of information on comprehensive metabolite profile and the maximum safe dose of the plant. This study aims at profiling the methanol leaf extract of T. catappa, quantifying total phenolic content (TPC) as well as the total flavonoid content (TFC) and evaluating its acute toxicity on blood, plasma biochemical parameters and histopathology of some vital organs in red hybrid tilapia (Oreochromis sp.). The experimental fish were acclimatised for 2 weeks and divided into six groups. Group (1) served as a control group and was administered 0.2 ml,g-1 of phosphate buffer saline (PBS). Groups 2-6 were orally administered T. catappa leaf extracts (0.2 ml.50 g-1 ) in the following sequence; 31.25, 62.5, 125, 250 and 500 mg.kg-1 body weight. The metabolites identified in T. catappa using liquid chromatography-tandem mass electrospray ionisation spectrometry (LC-ESI-MS/MS) revealed the presence of organic acids, hydrolysable tannins, phenolic acids and flavonoids. Phenolic quantification revealed reasonable quantity of phenolic compounds (217.48 µg GAEmg-1 for TPC and 91.90 µg. QCEmg-1 for TFC). Furthermore, there was no significant difference in all the tested doses in terms of blood parameters and plasma biochemical analysis except for the packed cell volume (PCV) at 500 mg.kg-1 when compared to the control. Significant histopathological changes were observed in groups administered with the extract at 125, 250 and 500 mg.kg-1 doses. To a very large extent it is therefore safe to administer the extract at 31.25 and 62.5 mg.kg-1 in tilapia.

1H NMR-Based Metabolomics Approach Revealing Metabolite Variation of Black Turmeric (Curcuma caesia) Extracts and Correlation with Its Antioxidant and α-Glucosidase Inhibitory Activities.

Research background: Curcuma species (Zingiberaceae) are well known medicinal herbs in India and Southeast Asia. Despite various findings reporting their beneficial biological activities, very little information has been recorded on the Curcuma caesia. Thus, this study aims to determine the phenolic content, antioxidant and α-glucosidase inhibitory activity of both rhizome and leaves of C. caesia. Experimental approach: Rhizome and leaves of C. caesia were dried with oven (OD) and freeze (FD)-drying methods, and extracted with different Φ(ethanol,water)=100:0, 80:20, 50:50 and 0:100. The bioactivities of C. caesia extracts were evaluated using in vitro tests; total phenolic content (TPC), antioxidant (DPPH and FRAP) and α-glucosidase inhibitory activity. Proton nuclear magnetic resonance (1H NMR)-based metabolomics approach was employed to differentiate the most active extracts based on their metabolite profiles and correlation with bioactivities. Results and conclusions: The FD rhizome extracted with Φ(ethanol,water)=100:0 was observed to have potent TPC expressed as gallic acid equivalents, FRAP expressed as Trolox equivalents and α-glucosidase inhibitory activity with values of (45.4±2.1) mg/g extract, (147.7±8.3) mg/g extract and (265.5±38.6) µg/mL (IC50), respectively. Meanwhile, for DPPH scavenging activity, the Φ(ethanol,water)=80:20 and 100:0 extracts of FD rhizome showed the highest activity with no significant difference between them. Hence, the FD rhizome extracts were selected for further metabolomics analysis. Principal component analysis (PCA) showed clear discrimination among the different extracts. Partial least square (PLS) analysis showed positive correlations of the metabolites, including xanthorrhizol derivative, 1-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-(6E)-6-heptene-3,4-dione, valine, luteolin, zedoardiol, ß-turmerone, selina-4(15),7(11)-dien-8-one, zedoalactone B and germacrone, with the antioxidant and α-glucosidase inhibition activities, whereas curdione and 1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-(lE,6E)-1,6-heptadiene3,4-dione were correlated with α-glucosidase inhibitory activity. Novelty and scientific contribution: C. caesia rhizome and leaf extracts contained phenolic compounds and had varies antioxidant and α-glucosidase inhibitory capacities. These findings strongly suggest that the rhizomes of C. caesia are an invaluable natural source of active ingredients for applications in pharmaceutical and food industries.

Nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry metabolomics studies on non-organic soybeans versus organic soybeans (Glycine max), and their fermentation by Rhizopus oligosporus.

BACKGROUND: Soybeans (Glycine max) are high in proteins and isoflavones, which offer many health benefits. It has been suggested that the fermentation process enhances the nutrients in the soybeans. Organic foods are perceived as better than non-organic foods in terms of health benefits, yet little is known about the difference in the phytochemical content that distinguishes the quality of organic soybeans from non-organic soybeans. This study investigated the chemical profiles of non-organic (G, T, U, UB) and organic (C, COF, A, R, B, Z) soybeans (G. max [L.] Merr.) and their metabolite changes after fermentation with Rhizopus oligosporus. RESULTS: A clear separation was only observed between non-organic G and organic Z, which were then selected for further investigation in the fermentation of soybeans (GF and ZF). All four groups (G, Z, GF, ZF) were analyzed using nuclear magnetic resonance (NMR) spectroscopy along with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this way a total of 41 and 47 metabolites were identified respectively, with 12 in common. A clear variation (|log1.5 FC| > 2 and P < 0.05) was observed between Z and ZF: most of the sugars and isoflavone glycosides were found only in Z, while more amino acids and organic acids were found in ZF. An additional four metabolites clustered as C-glycosylflavonoids were discovered from MS/MS-based molecular networking. CONCLUSION: Chemical profiles of non-organic and organic soybeans exhibited no significant difference. However, the metabolite profile of the unfermented soybeans, which were higher in sugars, shifted to higher amino acid and organic acid content after fermentation, thereby potentially enhancing their nutritional value. © 2022 Society of Chemical Industry.

LC-MS metabolomics analysis of Stevia rebaudiana Bertoni leaves cultivated in Malaysia in relation to different developmental stages.

INTRODUCTION: Stevia is known for its sweet taste, attributed to the presence of steviol glycosides. Although reports on the dynamic changes of steviol glycosides during development of stevia are available, the data are mainly focused on stevioside and rebaudioside A. Information concerning the comprehensive metabolite profile of stevia in relation to different developmental stages is still lacking. OBJECTIVE: This study investigated the metabolite changes along the developmental stages of a local stevia cultivar. METHODOLOGY: Stevia leaves were harvested at 4 different developmental stages (early vegetative, late vegetative, budding, and flowering). Samples were then subjected to LC-MS metabolomics analysis to determine the metabolite variations. RESULTS: A total of 55 metabolites, comprising phenolic acids, flavonoids, and terpenoids were identified by MS/MS analysis of the stevia leaf extracts, revealing a metabolite profile which was comparatively similar with those of cultivars grown in other countries. PLS-DA differentiated the early vegetative stage stevia leaf samples from those of the later stages by higher content of phenolic acids. The leaf metabolomes of the later 3 stages (late vegetative, budding, and flowering) were collectively richer in flavonoids. Meanwhile, the content of steviol glycosides is highest during the late vegetative and budding stages. CONCLUSION: The present study provided, for the first time, a general overview of the metabolite variations with regard to the different developmental stages of stevia. The information may facilitate decision making of suitable harvesting times for higher yields of steviol glycosides or a more balanced metabolite profile in terms of pharmacologically useful metabolites.

Complementary Analytical Platforms of NMR Spectroscopy and LCMS Analysis in the Metabolite Profiling of Isochrysis galbana.

This study was designed to profile the metabolites of Isochrysis galbana, an indigenous and less explored microalgae species. 1H Nuclear Magnetic Resonance (NMR) spectroscopy and Liquid Chromatography-Mass Spectrometry (LCMS) were used to establish the metabolite profiles of five different extracts of this microalga, which are hexane (Hex), ethyl acetate (EtOAc), absolute ethanol (EtOH), EtOH:water 1:1 (AqE), and 100% water (Aq). Partial least square discriminant analysis (PLS-DA) of the generated profiles revealed that EtOAc and Aq extracts contain a diverse range of metabolites as compared to the other extracts with a total of twenty-one metabolites, comprising carotenoids, polyunsaturated fatty acids, and amino acids, that were putatively identified from the NMR spectra. Meanwhile, thirty-two metabolites were successfully annotated from the LCMS/MS data, ten of which (palmitic acid, oleic acid, α-linolenic acid, arachidic acid, cholesterol, DHA, DPA, fucoxanthin, astaxanthin, and pheophytin) were similar to those present in the NMR profile. Another eleven glycerophospholipids were discovered using MS/MS-based molecular network (MN) platform. The results of this study, besides providing a better understanding of I.galbana's chemical make-up, will be of importance in exploring this species potential as a feed ingredient in the aquaculture industry.

In silico studies, nitric oxide, and cholinesterases inhibition activities of pyrazole and pyrazoline analogs of diarylpentanoids.

A new series of pyrazole, phenylpyrazole, and pyrazoline analogs of diarylpentanoids (excluding compounds 3a, 4a, 5a, and 5b) was pan-assay interference compounds-filtered and synthesized via the reaction of diarylpentanoids with hydrazine monohydrate and phenylhydrazine. Each analog was evaluated for its anti-inflammatory ability via the suppression of nitric oxide (NO) on IFN-γ/LPS-activated RAW264.7 macrophage cells. The compounds were also investigated for their inhibitory capability toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), using a modification of Ellman's spectrophotometric method. The most potent NO inhibitor was found to be phenylpyrazole analog 4c, followed by 4e, when compared with curcumin. In contrast, pyrazole 3a and pyrazoline 5a were found to be the most selective and effective BChE inhibitors over AChE. The data collected from the single-crystal X-ray diffraction analysis of compound 5a were then applied in a docking simulation to determine the potential binding interactions that were responsible for the anti-BChE activity. The results obtained signify the potential of these pyrazole and pyrazoline scaffolds to be developed as therapeutic agents against inflammatory conditions and Alzheimer's disease.

UHPLC-UV/PDA Method Validation for Simultaneous Quantification of Luteolin and Apigenin Derivatives from Elaeis guineensis Leaf Extracts: An Application for Antioxidant Herbal Preparation.

Luteolin and apigenin derivatives present in oil palm (Elaeis guineensis) leaves (OPL) are reported to possess excellent antioxidant properties relating to numerous health benefits. To meet the global demand for flavonoids, OPL, which is plentifully generated as an agricultural by-product from oil palm plantations, can be further exploited as a new source of natural antioxidant compounds. However, to produce a standardized herbal preparation, validation of the quantification method for these compounds is required. Therefore, in this investigation, we developed and validated an improved and rapid analytical method, ultra-high-performance liquid chromatography equipped with ultraviolet/photodiode array (UHPLC-UV/PDA) for the quantification of 12 luteolin and apigenin derivatives, particularly focusing on flavonoid isomeric pairs: orientin/isoorientin and vitexin/isovitexin, present in various OPL extracts. Several validation parameters were assessed, resulting in the UHPLC-UV/PDA technique offering good specificity, linearity, accuracy, precision, and robustness, where the values were within acceptable limits. Subsequently, the validated method was employed to quantify luteolin and apigenin derivatives from OPL subjected to different drying treatments and extraction with various solvent systems, giving total luteolin (TLC) and apigenin content (TAC) in the range of 2.04-56.30 and 1.84-160.38 µg/mg extract, respectively. Additionally, partial least square (PLS) analysis disclosed the combination of freeze dry-aqueous methanol yielded OPL extracts with high TLC and TAC, which are strongly correlated with antioxidant activity. Therefore, we provide the first validation report of the UHPLC-UV/PDA method for quantification of luteolin and apigenin derivatives present in various OPL extracts, suggesting that this approach could be employed in standardized herbal preparations by adopting orientin, isoorientin, vitexin, and isovitexin as chemical markers.

Clitorienolactones and Isoflavonoids of Clitorea ternatea Roots Alleviate Stress-Like Symptoms in a Reserpine-Induced Zebrafish Model.

Clitorea ternatea has been used in Ayurvedic medicine as a brain stimulant to treat mental illnesses and mental functional disorders. In this study, the metabolite profiles of crude C. ternatea root extract (CTRE), ethyl acetate (EA), and 50% aqueous methanol (50% MeOH) fractions were investigated using ultrahigh-performance liquid chromatography-diode array detector-tandem mass spectrometry (UHPLC-DAD-MS/MS), while their effect on the stress-like behavior of zebrafish, pharmacologically induced with reserpine, was investigated. A total of 32 compounds were putatively identified, among which, a series of norneolignans, clitorienolactones, and various flavonoids (flavone, flavonol, isoflavone, and isoflavanone) was found to comprise the major constituents, particularly in the EA and 50% MeOH fractions. The clitorienolactones, presently unique to the species, were present in both the free and glycosylated forms in the roots. Both the EA and 50% MeOH fractions displayed moderate effects on the stress-induced zebrafish model, significantly decreasing freezing duration and elevating the total distance travelled and average velocity, 72 h post-treatment. The results of the present study provide further evidence that the basis for the use of C. ternatea roots in traditional medicine to alleviate brain-related conditions, such as stress and depression, is attributable to the presence of clitorienolactones and the isoflavonoidal constituents.

2,4,6-Trihydroxy-3-geranyl acetophenone suppresses vascular leakage and leukocyte infiltration in lipopolysaccharide-induced endotoxemic mice.

CONTEXT: Lipopolysaccharide (LPS) exacerbates systemic inflammatory responses and causes excessive fluid leakage. 2,4,6-Trihydroxy-3-geranyl acetophenone (tHGA) has been revealed to protect against LPS-induced vascular inflammation and endothelial hyperpermeability in vitro. OBJECTIVE: This study assesses the in vivo protective effects of tHGA against LPS-induced systemic inflammation and vascular permeability in endotoxemic mice. MATERIALS AND METHODS: BALB/c mice were intraperitoneally pre-treated with tHGA for 1 h, followed by 6 h of LPS induction. Evans blue permeability assay and leukocyte transmigration assay were performed in mice (n = 6) pre-treated with 2, 20 and 100 mg/kg tHGA. The effects of tHGA (20, 40 and 80 mg/kg) on LPS-induced serum TNF-α secretion, lung dysfunction and lethality were assessed using ELISA (n = 6), histopathological analysis (n = 6) and survivability assay (n = 10), respectively. Saline and dexamethasone were used as the negative control and drug control, respectively. RESULTS: tHGA significantly inhibited vascular permeability at 2, 20 and 100 mg/kg with percentage of inhibition of 48%, 85% and 86%, respectively, in comparison to the LPS control group (IC50=3.964 mg/kg). Leukocyte infiltration was suppressed at 20 and 100 mg/kg doses with percentage of inhibition of 73% and 81%, respectively (IC50=17.56 mg/kg). However, all tHGA doses (20, 40 and 80 mg/kg) failed to prevent endotoxemic mice from lethality because tHGA could not suppress TNF-α overproduction and organ dysfunction. DISCUSSION AND CONCLUSIONS: tHGA may be developed as a potential therapeutic agent for diseases related to uncontrolled vascular leakage by combining with other anti-inflammatory agents.

α-Amylase and dipeptidyl peptidase-4 (DPP-4) inhibitory effects of Melicope latifolia bark extracts and identification of bioactive constituents using in vitro and in silico approaches.

CONTEXT: Melicope latifolia (DC.) T. G. Hartley (Rutaceae) was reported to contain various phytochemicals including coumarins, flavonoids, and acetophenones. OBJECTIVE: This study investigates the antidiabetic and antioxidant effects of M. latifolia bark extracts, fractions, and isolated constituents. MATERIALS AND METHODS: Melicope latifolia extracts (hexane, chloroform, and methanol), fractions, and isolated constituents with varying concentrations (0.078-10 mg/mL) were subjected to in vitro α-amylase and dipeptidyl peptidase-4 (DPP-4) inhibitory assay. Molecular docking was performed to study the binding mechanism of active compounds towards α-amylase and DPP-4 enzymes. The antioxidant activity of M. latifolia fractions and compounds were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and ß-carotene bleaching assays. RESULTS: Melicope latifolia chloroform extract showed the highest antidiabetic activity (α-amylase IC50: 1464.32 µg/mL; DPP-4 IC50: 221.58 µg/mL). Fractionation of chloroform extract yielded four major fractions (CF1-CF4) whereby CF3 showed the highest antidiabetic activity (α-amylase IC50: 397.68 µg/mL; DPP-4 IC50: 37.16 µg/mL) and resulted in ß-sitosterol (1), halfordin (2), methyl p-coumarate (3), and protocatechuic acid (4). Isolation of compounds 2-4 from the species and their DPP-4 inhibitory were reported for the first time. Compound 2 showed the highest α-amylase (IC50: 197.53 µM) and ß-carotene (88.48%) inhibition, and formed the highest number of molecular interactions with critical amino acid residues of α-amylase. The highest DPP-4 inhibition was exhibited by compound 3 (IC50: 911.44 µM). DISCUSSION AND CONCLUSIONS: The in vitro and in silico analyses indicated the potential of M. latifolia as an alternative source of α-amylase and DPP-4 inhibitors. Further pharmacological studies on the compounds are recommended.

Mass Spectrometry-Based Metabolomics Combined with Quantitative Analysis of the Microalgal Diatom (Chaetoceros calcitrans).

Although many metabolomics studies of higher land plant species have been conducted, similar studies of lower nonland plant species, which include microalgae, are still developing. The present study represents an attempt to characterize the metabolic profile of a microalgal diatom Chaetoceros calcitrans, by applying high-resolution mass spectrometry detection, via Q-ExactiveTM Plus Orbitrap mass spectrometry. The results showed that 54 metabolites of various classes were tentatively identified. Experimentally, the chloroform and acetone extracts were clearly distinguished from other solvent extracts in chemometric regression analysis using PLS, showing the differences in the C. calcitrans metabolome between the groups. In addition, specific metabolites were evaluated, which supported the finding of antioxidant and anti-inflammatory activities. This study also provides data on the quantitative analysis of four carotenoids based on the identification results. Therefore, these findings could serve as a reliable tool for identifying and quantifying the metabolome that could reflect the metabolic activities of C. calcitrans.

Comprehensive GCMS and LC-MS/MS Metabolite Profiling of Chlorella vulgaris.

The commercial cultivation of microalgae began in the 1960s and Chlorella was one of the first target organisms. The species has long been considered a potential source of renewable energy, an alternative for phytoremediation, and more recently, as a growth and immune stimulant. However, Chlorella vulgaris, which is one of the most studied microalga, has never been comprehensively profiled chemically. In the present study, comprehensive profiling of the Chlorella vulgaris metabolome grown under normal culture conditions was carried out, employing tandem LC-MS/MS to profile the ethanolic extract and GC-MS for fatty acid analysis. The fatty acid profile of C. vulgaris was shown to be rich in omega-6, -7, -9, and -13 fatty acids, with omega-6 being the highest, representing more than sixty percent (>60%) of the total fatty acids. This is a clear indication that this species of Chlorella could serve as a good source of nutrition when incorporated in diets. The profile also showed that the main fatty acid composition was that of C16-C18 (>92%), suggesting that it might be a potential candidate for biodiesel production. LC-MS/MS analysis revealed carotenoid constituents comprising violaxanthin, neoxanthin, lutein, ß-carotene, vulgaxanthin I, astaxanthin, and antheraxanthin, along with other pigments such as the chlorophylls. In addition to these, amino acids, vitamins, and simple sugars were also profiled, and through mass spectrometry-based molecular networking, 48 phospholipids were putatively identified.

Adsorption and Desorption Properties of Total Flavonoids from Oil Palm (Elaeis guineensis Jacq.) Mature Leaf on Macroporous Adsorption Resins.

Three different macroporous resins (XAD7HP, DAX-8, and XAD4) were evaluated for their adsorption and desorption properties in preparing flavonoid-enriched oil palm (Elaeis guineensis Jacq.) leaf extract. The influences of initial concentration, solution pH, contact time, and desorption solvent (ethanol) concentration were determined by static sorption/desorption methods. The optimal condition for adsorption of flavonoids was achieved when the solution of the extract was adjusted to pH 7, reaching equilibrium after 1440 min at 298 K. The adsorption process was well described by a pseudo-second-order kinetics model, while the adsorption isotherm data fitted well with a Freundlich model. The adsorption by each resin was via an exothermic and physical adsorption process. Based on the static experiment results, XAD7HP was found to be the most appropriate adsorbent, while 80% ethanol was the best solvent for desorbent. Further evaluation of its dynamic adsorption and desorption characteristics on a packed glass column showed that XAD7HP could enrich the OPL total flavonoid content by a 3.57-fold increment. Moreover, UHPLC-UV/PDA and UHPLC-MS/MS analysis revealed that apigenin and luteolin derivatives were selectively adsorbed by XAD7HP. Additionally, both the crude OPL extract and the flavonoid-enriched fraction have good DPPH and NO free radical scavenging activities. Multiple interactions between the flavonoids and cross-linked polymeric XAD7HP resin through van der Waals forces and hydrogen bonding described the sorption processes. Therefore, by utilizing this method, the flavonoid-enriched fraction from crude OPL extract could be used as a potential bioactive ingredient in nutraceutical and pharmaceutical applications at minimum cost with optimum efficiency.

Metabolite Characterization and Correlations with Antioxidant and Wound Healing Properties of Oil Palm (Elaeis guineensis Jacq.) Leaflets via 1H-NMR-Based Metabolomics Approach.

Oil palm (Elaeis guineensis Jacq.) leaflets (OPLs) are one of the major agricultural by-products generated from the massive cultivation of Malaysian palm oil. This biomass is also reported to be of potential value based on its health-improving effects. By employing proton nuclear magnetic resonance (1H-NMR) spectroscopy combined with multivariate data analysis (MVDA), the metabolite profile of OPLs was characterized and correlated with their antioxidant and wound healing properties. Principal component analysis (PCA) classified four varieties of extracts, prepared using solvents ranging from polar to medium polarity, into three distinct clusters. Cumulatively, six flavonoids, eight organic acids, four carbohydrates, and an amine were identified from the solvent extracts. The more polar extracts, such as, the ethyl acetate-methanol, absolute methanol, and methanol-water, were richer in phytochemicals. Based on partial least square (PLS) analysis, the constituents in these extracts, such as (+)-catechin, (-)-epicatechin, orientin, isoorientin, vitexin, and isovitexin, were strongly correlated with the measured antioxidant activities, comprising ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and nitric oxide (NO) free radical scavenging activities, as well as with cell proliferation and migration activities. This study has provided crucial evidence on the importance of these natural antioxidant compounds on the wound healing properties of OPL.

Identification of Dipeptidyl Peptidase-4 and α-Amylase Inhibitors from Melicope glabra (Blume) T. G. Hartley (Rutaceae) Using Liquid Chromatography Tandem Mass Spectrometry, In Vitro and In Silico Methods.

The present study investigated the antidiabetic properties of the extracts and fractions from leaves and stem bark of M. glabra based on dipeptidyl peptidase-4 (DPP-4) and α-Amylase inhibitory activity assays. The chloroform extract of the leaves was found to be most active towards inhibition of DPP-4 and α-Amylase with IC50 of 169.40 µg/mL and 303.64 µg/mL, respectively. Bioassay-guided fractionation of the leaves' chloroform extract revealed fraction 4 (CF4) as the most active fraction (DPP-4 IC50: 128.35 µg/mL; α-Amylase IC50: 170.19 µg/mL). LC-MS/MS investigation of CF4 led to the identification of trans-decursidinol (1), swermirin (2), methyl 3,4,5-trimethoxycinnamate (3), renifolin (4), 4',5,6,7-tetramethoxy-flavone (5), isorhamnetin (6), quercetagetin-3,4'-dimethyl ether (7), 5,3',4'-trihydroxy-6,7-dimethoxy-flavone (8), and 2-methoxy-5-acetoxy-fruranogermacr-1(10)-en-6-one (9) as the major components. The computational study suggested that (8) and (7) were the most potent DPP-4 and α-Amylase inhibitors based on their lower binding affinities and extensive interactions with critical amino acid residues of the respective enzymes. The binding affinity of (8) with DPP-4 (-8.1 kcal/mol) was comparable to that of sitagliptin (-8.6 kcal/mol) while the binding affinity of (7) with α-Amylase (-8.6 kcal/mol) was better than acarbose (-6.9 kcal/mol). These findings highlight the phytochemical profile and potential antidiabetic compounds from M. glabra that may work as an alternative treatment for diabetes.

Pharmacokinetics and Metabolism of Liposome-Encapsulated 2,4,6-Trihydroxygeranylacetophenone in Rats Using High-Resolution Orbitrap Liquid Chromatography Mass Spectrometry.

2,4,6-trihydroxy-3-geranylacetophenone (tHGA) is a bioactive compound that shows excellent anti-inflammatory properties. However, its pharmacokinetics and metabolism have yet to be evaluated. In this study, a sensitive LC-HRMS method was developed and validated to quantify tHGA in rat plasma. The method showed good linearity (0.5-80 ng/mL). The accuracy and precision were within 10%. Pharmacokinetic investigations were performed on three groups of six rats. The first two groups were given oral administrations of unformulated and liposome-encapsulated tHGA, respectively, while the third group received intraperitoneal administration of liposome-encapsulated tHGA. The maximum concentration (Cmax), the time required to reach Cmax (tmax), elimination half-life (t1/2) and area under curve (AUC0-24) values for intraperitoneal administration were 54.6 ng/mL, 1.5 h, 6.7 h, and 193.9 ng/mL·h, respectively. For the oral administration of unformulated and formulated tHGA, Cmax values were 5.4 and 14.5 ng/mL, tmax values were 0.25 h for both, t1/2 values were 6.9 and 6.6 h, and AUC0-24 values were 17.6 and 40.7 ng/mL·h, respectively. The liposomal formulation improved the relative oral bioavailability of tHGA from 9.1% to 21.0% which was a 2.3-fold increment. Further, a total of 12 metabolites were detected and structurally characterized. The metabolites were mainly products of oxidation and glucuronide conjugation.

Subacute Oral Administration of Clinacanthus nutans Ethanolic Leaf Extract Induced Liver and Kidney Toxicities in ICR Mice.

This study investigated the leaves of Clinacanthus nutans for its bioactive compounds and acute and subacute toxicity effects of C. nutans ethanolic leaf extract (CELE) on blood, liver and kidneys of ICR mice. A total of 10 8-week-old female mice were divided into groups A (control) and B (2000 mg/kg) for the acute toxicity study. A single dose of 2000 mg/kg was administered to group B through oral gavage and mice were monitored for 14 days. In the subacute toxicity study, mice were divided into five groups: A (control), B (125 mg/kg), C (250 mg/kg), D (500 mg/kg) and E (1000 mg/kg). The extract was administered daily for 28 days via oral gavage. The mice were sacrificed, and samples were collected for analyses. Myricetin, orientin, isoorientin, vitexin, isovitexin, isookanin, apigenin and ferulic acid were identified in the extract. Twenty-eight days of continuous oral administration revealed significant increases (p < 0.05) in creatinine, ALT and moderate hepatic and renal necrosis in groups D and E. The study concluded that the lethal dose (LD50) of CELE in mice is greater than 2000 mg/kg and that repeated oral administrations of CELE for 28 days induced hepatic and renal toxicities at 1000 mg/kg in female ICR mice.

Variation in the metabolites and α-glucosidase inhibitory activity of Cosmos caudatus at different growth stages.

BACKGROUND: Cosmos caudatus is an annual plant known for its medicinal value in treating several health conditions, such as high blood pressure, arthritis, and diabetes mellitus. The α-glucosidase inhibitory activity and total phenolic content of the leaf aqueous ethanolic extracts of the plant at different growth stages (6, 8. 10, 12 and 14 weeks) were determined in an effort to ascertain the best time to harvest the plant for maximum medicinal quality with respect to its glucose-lowering effects. METHODS: The aqueous ethanolic leaf extracts of C. caudatus were characterized by NMR and LC-MS/MS. The total phenolic content and α-glucosidase inhibitory activity were evaluated by the Folin-Ciocalteu method and α-glucosidase inhibitory assay, respectively. The statistical significance of the results was evaluated using one-way ANOVA with Duncan's post hoc test, and correlation among the different activities was performed by Pearson's correlation test. NMR spectroscopy along with multivariate data analysis was used to identify the metabolites correlated with total phenolic content and α-glucosidase inhibitory activity of the C. caudatus leaf extracts. RESULTS: It was found that the α-glucosidase inhibitory activity and total phenolic content of the optimized ethanol:water (80:20) leaf extract of the plant increased significantly as the plant matured, reaching a maximum at the 10th week. The IC50 value for α-glucosidase inhibitory activity (39.18 µg mL- 1) at the 10th week showed greater potency than the positive standard, quercetin (110.50 µg mL- 1). Through an 1H NMR-based metabolomics approach, the 10-week-old samples were shown to be correlated with a high total phenolic content and α-glucosidase inhibitory activity. From the partial least squares biplot, rutin and flavonoid glycosides, consisting of quercetin 3-O-arabinofuranoside, quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, and quercetin 3-O-xyloside, were identified as the major bioactive metabolites. The metabolites were identified by NMR spectroscopy (J-resolve, HSQC and HMBC experiments) and further supported by dereplication via LC-MS/MS. CONCLUSION: For high phytomedicinal quality, the 10th week is recommended as the best time to harvest C. caudatus leaves with respect to its glucose lowering potential.